Dominant negative isoform of the Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia.

Gene targeting studies in mice have shown that the transcription factor Ikaros plays an essential role in lymphoid development and as a tumor suppressor in T cells, whereas the related gene Aiolos functions as a tumor suppressor in B cells. We analyzed the expression levels of the Ikaros gene family, Ikaros and Aiolos, in human bone marrow samples from patients with adult acute lymphoblastic leukemia [ALL (n = 46; B-cell ALL = 41; T-cell ALL = 5)]. Overexpression of the dominant negative isoform of Ikaros gene Ik-6 was observed in 14 of 41 B-cell ALL patients by reverse transcription-PCR, and the results were confirmed by sequencing analysis and immunoblotting. None of the other dominant negative isoforms of the Ikaros gene were detected by reverse transcription-PCR analysis. Southern blotting analysis with PstI digestion revealed that those patients with the dominant negative isoform Ik-6 might have small mutations in the Ikaros locus. We did not detect any overexpression of dominant negative isoforms of Aiolos in adult ALL patients. These results suggest that Ikaros plays a key role in human B-cell malignancies through the dominant negative isoform Ik-6.

Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.

Gene targeting studies in mice have shown that the lack of Ikaros activity leads to T-cell hyperproliferation and T-cell neoplasia, establishing the Ikaros gene as a tumor suppressor gene in mice. This prompted us to investigate whether mutations in Ikaros play a role in human hematological malignancies. Reverse transcription-PCR was used to determine the relative expression levels of Ikaros isoforms in a panel of human leukemia/lymphoma cell lines and human bone marrow samples from patients with hematological malignancies. Among the cell lines examined, only BV-173, which was derived from a chronic myelogenous leukemia (CML) patient in lymphoid blast crisis, overexpressed the dominant-negative isoform, Ik-6. In 9 of 17 samples of patients in blast crisis of CML, Ikaros activity had been reduced either by drastically reducing mRNA expression (4 of 17) or by overexpressing the dominant-negative isoform Ik-6 (5 of 17). Significantly, expression of Ikaros isoforms seemed normal in chronic phase CML patients and patients with other hematological malignancies. In some cases, overexpression of the dominant-negative Ik-6 protein was confirmed by Western blot analysis, and Southern blot analysis indicated that decreases in Ikaros activity correlated with a mutation in the Ikaros locus. In summary, these findings suggest that a reduction of Ikaros activity may be an important step in the development of blast crisis in CML and provide further evidence that mutations that alter Ikaros expression may contribute to human hematological malignancies.

Ikaros sets thresholds for T cell activation and regulates chromosome propagation.

T cell activation involves the sustained accumulation of T cell receptor (TCR) and IL-2 receptor (IL-2R) mediated signaling events that promote cell cycle entry and progression. The Ikaros family of nuclear factors regulate this process by providing thresholds overcome by receptor signaling. T cells with reduced levels of Ikaros activity require fewer TCR engagement events for activation, exhibit a greater proliferative response to IL-2, and are less sensitive to inhibitors of TCR and IL-2R signaling. Upon T cell activation, Ikaros proteins localize in a higher-order chromatin structure where they colocalize with components of the DNA replication machinery. Proliferating T cells with reduced Ikaros activity display chromosome abnormalities. We propose that participation of Ikaros in higher-order chromatin structures controls cell cycle transitions and restricts DNA replication.

Aiolos regulates B cell activation and maturation to effector state.

Aiolos encodes a zinc finger DNA-binding protein that is highly expressed in mature B cells and is homologous to Ikaros. In the periphery of mice homozygous for an Aiolos-null mutation, B cells exhibit an activated cell surface phenotype and undergo augmented antigen receptor (BCR)-mediated in vitro proliferative responses, even at limiting amounts of stimulant. In vivo, T cell-dependent B cell responses, including the formation of germinal centers and elevated serum IgG and IgE, are detected in Aiolos-deficient mice in the absence of immunization. Auto-antibodies and development of B cell lymphomas are frequently seen among aging Aiolos mutants. In sharp contrast to conventional B cells, B cells of the peritoneum, of the marginal zone, and the recirculating bone marrow population are greatly reduced.

Helios, a novel dimerization partner of Ikaros expressed in the earliest hematopoietic progenitors.

BACKGROUND: Normal hematopoietic development depends on the activity of the Ikaros transcription factor, which contains distinct zinc-finger domains that mediate DNA binding and protein dimerization. Mice homozygous for a transgene encoding a dominant-negative version of Ikaros that lacks the DNA-binding domain but not the dimerization domain have a more severe phenotype than Ikaros null mice. This observation suggests the presence of factor(s) that can dimerize with Ikaros and partially complement its function. One previously identified factor, Aiolos, probably serves this role in the lymphoid system; a related factor involved in hematopoietic progenitors remains unknown, however. RESULTS: Here, we describe the cloning of an Ikaros-related gene, Helios. Analysis of the primary sequences of Helios, Ikaros and Aiolos revealed that the DNA-binding, transcriptional activation and dimerization domains are functionally conserved. Helios activated transcription from Ikaros DNA-binding sites and could dimerize with itself, Ikaros or Aiolos. Expression of Helios was detected in the earliest hematopoietic sites of the embryo, in hematopoietic stem cells in the adult and was subsequently restricted to a subset of cells in the T cell lineage. Helios co-localized with Ikaros and Aiolos proteins in macromolecular nuclear structures and formed stable complexes in vivo with the dominant-negative version of Ikaros. CONCLUSIONS: Distinct but overlapping expression patterns of members of the Ikaros gene family during hematopoiesis might result in the formation of different multimeric complexes that have specific roles in lineage progression. The preferential expression of Helios in the earliest stages of hematopoiesis suggests that this gene functions predominantly in early progenitors.

Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation.

Development of the lymphoid system is dependent on the activity of zinc finger transcription factors encoded by the Ikaros gene. Differences between the phenotypes resulting from a dominant-negative and a null mutation in this gene suggest that Ikaros proteins act in concert with another factor with which they form heterodimers. Here we report the cloning of Aiolos, a gene which encodes an Ikaros homologue that heterodimerizes with Ikaros proteins. In contrast to Ikaros--which is expressed from the pluripotent stem cell to the mature lymphocyte--Aiolos is first detected in more committed progenitors with a lymphoid potential and is strongly up-regulated as these differentiate into pre-T and pre-B cell precursors. The expression patterns of Aiolos and Ikaros, the relative transcriptional activity of their homo- and heteromeric complexes, and the dominant interfering effect of mutant Ikaros isoforms on Aiolos activity all strongly suggest that Aiolos acts in concert with Ikaros during lymphocyte development. We therefore propose that increasing levels of Ikaros and Aiolos homo- and heteromeric complexes in differentiating lymphocytes are essential for normal progression to a mature and immunocompetent state.

The role of the Ikaros gene in lymphocyte development and homeostasis.

The Ikaros gene, which encodes a family of hemopoietic-specific zinc finger proteins, is described as a central regulator of lymphocyte differentiation. During fetal development, it is required at the earliest stage of T cell and B cell specification. In the adult, however, lymphoid lineages rely on Ikaros at distinct phases of their development. Its activity is essential for the generation of B cell but not of T cell precursors, although the differentiation of the latter is not normal. A significant increase in CD4 thymocytes and their immediate precursors is detected, and because these cells lack markers that correlate with positive selection, a deregulation in their maturation process is suggested. Furthermore, Ikaros-null thymocytes hyperproliferate in response to T cell receptor (TCR) signaling; within days after their appearance in the thymus, clonally expanding populations are detected. Deregulated TCR-mediated responses and the fast kinetics of tumor development in these mutant thymocytes implicate Ikaros as a central tumor suppressor gene for the T cell lineage. In addition, lack of natural killer cells and selective defects in gamma delta T cells and dendritic antigen-presenting cells point to Ikaros as an essential factor for the establishment of early branchpoints of the T cell pathway. The dominant interference activity of Ikaros isoforms unable to bind DNA and their effects in lymphocyte development suggest that Ikaros works in concert with other factors. The role of Aiolos, a lymphoid-restricted and structurally related gene, in lymphoid differentiation is discussed. A model is proposed that defines Ikaros as the backbone of a complex regulatory protein network that controls cell fate decisions and regulates homeostasis in the hemo-lymphoid system. Changes in this regulatory network may reflect differentiation and proliferation adjustments made in hemo-lymphoid progenitors and precursors as they give rise to the cells of our immune system.

A 125 bp region of the Ig VH1 promoter is sufficient to confer lymphocyte-specific expression in transgenic mice.

Ig variable region (VH) promoters are active only in B cells and extinction experiments suggest that they are negatively regulated in non-B lineage cells. In contrast to the multiple transcription factor binding sites which occur in Ig enhancers, only a few functionally important transcription factor binding sites have been identified in VH promoters. In this study, we have used transgenic animals to test the functional importance of a 5' portion of the VH1 promoter which is known to contain a matrix attachment region as well as binding sites for the negative regulator NF-microNR and Bright, a protein complex which is induced upon stimulation of B cells with IL-5 and antigen. Our results show that none of these regions is required for VH1 promoter activity in the context of a rearranged micro gene. In fact, a truncated promoter extending only 125 bp upstream of the transcription initiation site was sufficient to confer strong expression in lymphocytes with negligible expression in any other tissue. These results define the smallest known region of a VH promoter which is capable of lymphoid-specific activity and establish the simplicity of the VH1 promoter element.

Ig/EBP (C/EBP gamma) is a transdominant negative inhibitor of C/EBP family transcriptional activators.

Analysis of cDNA and genomic clones shows that the murine Ig/EBP (C/EBP gamma) gene encodes a small protein with a predicted molecular weight of 16.4 kDa which contains C/EBP family basic and leucine zipper domains but lacks the transcriptional activation domains present in C/EBP (C/EBP alpha) and NF-IL6 (C/EBP beta). In transfection assays Ig/EBP is neither an activator nor a repressor of transcription; however, Ig/EBP inhibits the transcriptional ability of NF-IL6 (C/EBP beta) and C/EBP (C/EBP alpha), acting as a transdominant negative regulator. Thus Ig/EBP resembles LIP, another negative regulator of the C/EBP family, in both structure and transcriptional activity. Of the three known C/EBP family inhibitors, Ig/EBP, LIP and CHOP-10, only Ig/EBP is ubiquitously expressed. Therefore, Ig/EBP may act as a general buffer for C/EBP activators in many cell types.

TFE3 contains two activation domains, one acidic and the other proline-rich, that synergistically activate transcription.

TFE3 is a basic-helix-loop-helix-zipper (bHLHZIP) domain-containing protein that binds mu E3 sites in regulatory elements in the immunoglobulin heavy chain gene. The protein is a transcriptional activator that is expressed in vivo as two alternately spliced isoforms with different activating properties: TFE3L contains an N-terminal acidic activation domain; TFE3S lacks this activation domain and is a dominant negative inhibitor of TFE3L. We show that TFE3L and TFE3S contain a second, C-terminal activation domain rich in proline residues. This pro-rich activation domain has activity in a Gal4 fusion assay comparable to the N-terminal acidic activation domain present in TFE3L. The TFE3 pro-rich activation domain contains regions of strong homology with the related proteins microphthalmia and TFEB, suggesting that these regions are important for function. Using two different assays, we show that the N- and C-terminal activation domains of TFE3 act synergistically. This synergism explains in part the ability of TFE3S to act as a dominant negative. Our domain analysis of TFE3 is incorporated into a general structural model for the TFE3 protein that predicts that the activation domains of TFE3 will be widely separated in space.

The C/EBP family of proteins distorts DNA upon binding but does not introduce a large directed bend.

The DNA-bending properties of several C/EBP family proteins, bound to two different sites, have been determined using circular permutation and phasing analyses. All the proteins examined by circular permutation analysis induced a significant distortion in the DNA (40-66 degrees), and this distortion was of the same magnitude at both C/EBP sites. However, phasing analysis revealed that the size of the directed bend induced in the DNA by C/EBP proteins was only 1-4 degrees. Both the magnitude and orientation of the directed bend were affected by the specific sequence of the DNA-binding site. All proteins induced a directed bend of similar magnitude and orientation (toward the minor groove) when bound to the C/EBP site derived from the IgH enhancer, but the induced bend was smaller when the protein was bound to the C/EBP site derived from the VH1 promoter. The study also included a heterodimer between Ig/EBP and ATF4. Similar to C/EBP protein homodimers, this heterodimer induces a small directed bend of 4 degrees oriented toward the minor groove when bound to the enhancer-derived C/EBP site.

The C/EBP family of transcriptional activators is functionally important for Ig VH promoter activity in vivo and in vitro.

We have examined the functional importance of binding sites for C/EBP family members (E sites), in two Ig VH promoters: VH1, a member of the S107 family, and BCL1, a member of the J558 family. Mutation of the E site in the VH1 promoter diminishes transcription in vivo to 59% of wild-type and transcription from the BCL1 promoter in vitro is inhibited to an average of 39% of wild-type by competition with E site oligonucleotides. Purified E site binding proteins from plasmacytoma cells stimulated BCL1 transcription in vitro 2.3-fold. Although five C/EBP family proteins are known which bind the E site, antibody ablation of DNA:protein complexes resolved by electrophoretic mobility shift assays showed that Ig/EBP-1 is the only E site binding protein detectable in early B cell lines; more mature B cells contain Ig/EBP-1 and NF-IL6. We also show by antibody-depletion that Ig/EBP-1 activates the BCL1 promoter in vitro.